VDAC-tubulin Regulation of Mitochondrial Membrane Potential Heterogeneity Heterogeneity of mitochondrial ??, an overall indicator of mitochondrial metabolism, is a well- recognized and very poorly understood phenomenon in cancer. Cancer bioenergetics is characterized by enhanced glycolysis (Warburg phenotype) and partial suppression of mitochondrial metabolism. Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, pumps protons to the intermembrane space creating a negative mitochondrial membrane potential (??), and produces reactive oxygen species (ROS). OXPHOS depends on the ingress of respiratory substrates through Voltage Dependent Anion Channels (VDAC) located in the mitochondrial outer membrane. Free tubulin promotes VDAC closure and suppresses mitochondrial metabolism contributing to the Warburg effect. Relevant to this application, the inhibitory effect of tubulin on VDAC is enhanced by protein kinase A (PKA)-dependent VDAC phosphorylation and antagonized by the small molecule erastin. The central hypothesis of this proposal is that heterogeneity of ?? in cancer cells, indicative of heterogeneity in mitochondrial and Warburg metabolism, is largely dependent on VDAC opening. We propose that VDAC in low vs high ?? cells is regulated by two main factors: the type of VDAC-isoform/?-tubulin isotype interaction and the PKA-dependent VDAC phosphorylation. Accordingly, in Specific Aim 1, we will determine mRNA levels and protein expression of different VDAC isoforms and ?-tubulin isotypes in high and low ?? human hepatocarcinoma cells. To characterize different types of VDAC-tubulin interactions we will use proximity ligation assays, colocalization by immunocytochemistry and immunoprecipitation. In Specific Aim 2, independent of mechanisms causing heterogeneity, we will determine constitutive intercellular and intracellular distribution of ??, NADH and redox state in intact cells using advanced quantitative confocal microscopy and 3-D reconstructions. In addition, we will assess respiration, glycolytic flux, ROS generation and cell proliferation and migration in high vs low ?? cells in the presence or absence of PKA activation/inhibition and erastin-dependent VDAC opening. Proliferating non-cancerous human embryonic stem cells and differentiated human hepatocytes will be used for comparison. Overall, this project will elucidate if constitutive heterogeneity of ?? is contributed by specific VDAC isoforms/?-tubulin interactions, if it indicates a predominant oxidative or glycolytic phenotype and if it correlates with heterogeneity in ROS formation. Further, it will also assess if constitutive ?? is an indicator of metabolic fitness for cell proliferation.